Insecticidal properties of pyrethrum have been known for many years, dating back to folk entomology and has been safely used, handled, and dispersed in the home and around pets, people, domestic animals and food plants. Pyrethrum is the name applied to compounds produced by Chrysanthemum cinerariaefolium and Chrysanthemum coccineum, plants which produce and store within the oil glands of the flowers six closely related esters known as pyrethrins (see FIG. 1), the most potent of which are pyrethrins I and II. When extracted, these pyrethrin compounds are nonpersistent insecticides with relatively low toxicity to mammals. The term pyrethrum is also used to refer to the commercial extract from pyrethrin flowers which contains the pyrethrin esters.
Industrially, pyrethrum extracts are obtained by extraction of dried pyrethrum flowers with hexane followed by dewaxing and decolorization to yield a mixture containing approximately 20% pyrethrins and 80% inert plant materials or solvents. This technical extract is registered with the Environmental Protection Agency and is a standard item of commerce used for formulating numerous end products. Flower production is centered in Kenya and surrounding countries, with some production being attempted in Tasmania and New Guinea. While pyrethrum flowers are not grown commercially in the United States, some of the Compositae (daisies, marigolds, etc.) in U.S. gardens probably produce these compounds. There has been an effort to cultivate C. cinerariaefolium in Oregon and Arizona but this is not yet a viable commercial source.
Recent trends toward the use of pesticides with greater environmental safety are causing an increase in pyrethrum demand, but the supply has remained relatively constant. Market demand exceeds supply by more than four times, thus generating a need to find an inexpensive method for chemically producing pyrethrins.
Synthetic pyrethroids have been developed which are more efficacious and longer lasting than pyrethrin, but these improvements are accompanied by increased toxicity and persistence of toxic residues. Compared with synthetic pyrethroids, pyrethrin has the favorable characteristic of being highly efficacious on a broad spectrum of insects while still being environmentally safe. To increase the supply of pyrethrins, the extraction/isolation methods currently used can be improved in such factors as (1) enhanced yield (2) decreased contamination by inert plant material and (3) delivery of a specific ratio of the six pyrethrins. Alternatively, pyrethrins can be chemically synthesized. To obtain optimal activity from a synthetic pyrethrin, the molecule(s) must have the correct three-dimensional spatial configuration (stereochemistry) because the incorrect stereoisomers are significantly less active as insecticides. Although stereospecific synthesis of pyrethrins utilizing synthetic organic methods is possible, the cost is prohibitive which causes continued reliance on extracts from flowers. Reactions contemplated in the synthesis of pyrethrins are schematically presented in FIGS. 2 and 3.
Pyrethrin molecules are composed of two distinct parts, a substituted cyclopropyl carboxylic acid and an unsaturated keto-alcohol entity. The cyclopropyl carboxylic acid part of the molecule, chrysanthemic or pyrethric acid, possesses complex stereochemistry. Chrysanthemic acid contains two asymmetric centers with the possibility of yielding four isomers. Pyrethric acid contains the same asymmetric centers and additionally has the possibility of cis-trans isomerism in the vinyl side chain to yield eight possible isomers.
The absolute configuration of the cyclopropane part of the pyrethrate esters, (+)-trans-chrysanthemic acid and (+)-trans-trans-pyrethric acid, have been determined chemically [Crombie, L. (1954) J. Chem. Soc., London, 470], and spectroscopically, [Begley, M. J. et al. (1972) J. Chem. Soc., Chem. Commun., 1276]. The naturally occurring configurations are insecticidally more active than the racemic mixture or any of the synthetic isomers. Because of the complex stereochemistry and thus difficulty in preparing chrysanthemic acid stereo- and enantiospecifically, a biological approach to this material is attractive as an economically feasible alternative.
Rethrolones, which is the collective name applied to the keto-alcohol components of the insecticidal pyrethrin esters, are a group of closely related cyclopentenones. Some studies have indicated that the rethrolone portion of the molecule is synthesized through a pathway different than chrysanthemic acid since label from .sup.14 C-acetate is found on the rethrolone portion of the molecule, but not from .sup.14 C-mevalonate. Although specific information is not available, it has been suggested that fatty acids, such as linoleic acid, are biosynthetic intermediates of the rethrolone portion of pyrethrin molecules, [Vick, B. A. et al. (1984) Plant Physiol. 75:458; Vick, B. A. et al (1987) Plant Physiol. 85:1073; Crombie, L. et al. (1985) Perkin Trans. I, p. 1393; Hildebrand, D. F. (1989) Physiol. Plant 76:249].
The rethrolone keto-alcohols have one asymmetric carbon at the 4-position and a double bond(s) in the side chain which is capable of cis-trans isomerism in the 2-position. It is therefore possible to have up to four stereoisomers for each keto-alcohol. It has been shown that only the (+) form occurs in the natural esters and that the natural configurations are insecticidally more active, [Elliot, M. et al. (1971) J. Chem. Soc., p. 2548; Katsuda, Y. et al. (1958) Bull. Agr. Chem. Soc. Japan 22:427].
It has been established that biosynthesis of the cyclopropane moiety of pyrethrin molecules, chrysanthemic or pyrethric acid, stems from mevalonic acid as the starting material. [Pattenden, G. et al. (1973) Tetrahedron Lett. 36:3473; Abou-Donia, S. A. et al. (1973) Tetrahedron Lett. 36:3477; Crombie, L. (1980) Pestic Sci. 11:102; Pattenden, G. (1970) Pyrethrum Post 10(4):2; Levy, L. W. et al. (1960) Pyrethrum Post 5(4):3].
Reactions 1 to 4 in FIG. 2 occur in all organisms which convert mevalonic acid to dimethylallyl pyrophosphate (DMAPP), the proposed 5-carbon precursor to chrysanthemyl diphosphate). The enzymes responsible for these reactions have been purified. Isopentenyl pyrophosphate (also called isopentenyl diphosphate) and DMAPP are the precursors to many metabolites including sterols, pigments and certain vitamins. The enzyme responsible for the interconversion of isopentenyl diphosphate and DMAPP (reaction 4) is isopentenyl pyrophosphate isomerase (EC 5.3.3.2) which has been purified from various animals and bacteria, [Satterwhite, D. M. (1985) Methods Enzymol. 110:92), and plants such as Capsicum, Dogbo, O. et al. (1987) Biochim. Biophys. Acta 920:140; daffodils, Lutzow, M. et al. (1988) Biochim. Biophys. Acta 959:118]; and tomato, Spurgeon, S. L. et al. (1984) Arch. Biochem. Biophys. 230(2):446-454].
Chrysanthemic acid has been reported to be synthesized in certain members of the Compositae family, including species of Chrysanthemum, Tagetes, Artemisia and Santolina, [Zito, S. W. et al. (1985) U.S. Pat. No. 4,525,455, Banthorpe et al. (1977) Phytochemistry 16:85]. It was suggested [Casida, J. E. (1973) "Pyrethrum, The Natural Insecticide," Casida, ed., Academic Press, New York, pp. 101-120; Manitto, P. (1981) "Biosynthesis of Natural Products," Wiley & Sons, New York, pp . 94-95], that two molecules of DMAPP condense to produce chrysanthemyl diphosphate (also called chrysanthemyl pyrophosphate; reaction 5 in FIG. 2). Although the exact mechanism of this novel proposed combination of C-5 units is unknown, it was suggested to be a "non-head-to-tail" condensation and thus was unlike the reactions which lead to the synthesis of most other isoprenoids. There are no reports on the purification of the enzyme responsible for the formation of chrysanthemyl diphosphate, so it has not been established that DMAPP is the immediate precursor for this intermediate. Zito and Staba (U.S. Pat. No. 4,525,455) have demonstrated that both .sup.14 C-mevalonic acid and .sup.14 C-isopentenyl diphosphate produce .sup.14 C-chrysanthemyl diphosphate using a cell-free homogenate from C. cinerariaefolium. Using crude enzyme homogenates from Artemisia annua and Santolina chamaecyparissus, Banthorpe, D. V. (1977) Phytochemistry 16:85 demonstrated that .sup.14 C-DMAPP and .sup.14 C-dimethylvinylcarbinol were converted to chrysanthemyl diphosphate. The work by Zito and Stabe, supra indicates that the enzymes forming chrysanthemyl diphosphate and the other enzymes involved in pyrethrin formation are cytosolic, since essentially all activity was found in the 100,000 X g supernatant and little to no activity was observed in the proplastid and mitochondrial fractions.
The enzyme responsible for the putative condensation reaction to form chrysanthemyl diphosphate from DMAPP is designated chrysanthemyl diphosphate synthase (CDS) and is a prenyltransferase. Prenyltransferases (EC 2.5.1.x) catalyze the transfer of an isoprenoid diphosphate to another isoprenoid diphosphate or to a nonisoprenoid acceptor. That only one enzyme could be involved in the conversion of DMAPP to chrysanthemyl diphosphate is analogous to the enzymatic function of a different prenyltransferase which, even when purified to homogeneity, exhibits two catalytic activities, (Dogbo, O. et al. (1988) Proc. Natl. Acad. Sci. USA 85:7054). This prenyltransferase protein, designated phytoene synthase, couples two molecules of geranylgeranyl diphosphate to yield prephytoene diphosphate and then converts prephytoene diphosphate into phytoene. Similarly, squalene synthetase in the presence of Mg.sup.2+ and a reduced pyridine nucleotide, forms squalene by a two step reductive condensation of two molecules of farnesyl diphosphate.
Pyrethroid refers to a non-naturally occurring insecticidal compound chemically similar to a pyrethrin molecule. Some of the earliest pyrethroids chemically synthesized were simply substituted benzyl esters of chrysanthemic acid having insecticidal activity, for example, 4-allylbenzyl chrysanthemate. In 1965, the ester of chrysanthemic acid and 5-benzyl-3-furylmethyl alcohol, named resmethrin, was synthesized and found to be about twenty times as active as pyrethrin I against houseflies. Bioresmethrin, (+)-trans-resmethrin, is the most active of several isomers that make up the mixture constituting resmethrin. Bioresmethrin is twice as active as resmethrin and about fifty times as active as DDT [1,1,1-trichloro-2,2-bis-(p-chloro-phenyl)ethane] against houseflies.
In the 1970s three pyrethroids, permethrin, cypermethrin and decamethrin (deltamethrin), were chemically synthesized and were found to have high stability and high intrinsic insecticidal activity. These compounds were produced by varying the alcohol moiety and adding halogen substituents onto the side chains of the chrysanthemic acid part of the molecule. Introducing an .alpha.-cyano group approximately trebled the activity, which was further enhanced with dihalovinyl substituents and the side chain cis rather than trans to the ester group. These early beginnings in the synthesis of active pyrethroids quickly led to much competition in the search for new pyrethroids and many companies now have research and development programs in this field.
The currently available methods for the synthesis of pyrethroids generally produce mixtures of all possible isomers, each of which has a different level of biological activity. Usually, the product is marketed as a racemic mixture. Additionally, the different isomers are separated to varying extents depending on the resolution capability of the available technology, and the resolved forms of a pyrethroid are also marketed. Only in one case is a pyrethroid commercially available as a single active isomeric species. For example, deltamethrin is the only pyrethroid, to date, that is marketed as a single isomer, i.e., the most active D-cis isomer.